This study was designed to investigate the effects of supplementation of spent mushroom substrates (SMS) on rumen fermentation and blood metabolites in Hanwoo steers. The experiment was conducted as a duplicated Latin square design with six Hanwoo steers (), each permanently fitted with a ruminal cannula. There were three treatments; i) control (concentrates 4.8 kg +rice straw 1.2 kg/d), ii) Pleurotus eryngiia (PE) treatment (concentrates 4.8 kg+rice straw 0.73 kg+Pleurotus eryngiia 1.20 kg/d) and iii) Pleurotus osteratus (PO) treatment (concentrates 4.8 kg+rice straw 0.73 kg+Pleurotus osteratus 1.20 kg/d). There were no major effects of different dietary treatments on rumen parameters such as pH, ammonia-N, individual and total VFA production. Parameters of N utilization, including blood urea nitrogen (BUN), total protein and albumin levels, were not significantly different among the treatments, except for creatinine. Thus, the present results indicated that protein utilization was mostly unaffected by SMS treatments such as PE and PO, even though creatinine concentration was lower in PE compared with control and PO treatments (p<0.05). The present results indicate that Pleurotus eryngii and Pleurotus osteratus could be used as a forage source to replace 40% of rice straw without any negative effects on rumen fermentation and blood metabolites in Hanwoo steers.